ATP-dependent enzymes were investigated as to the stringency of their ATP requirement. For all the enzymes examined except firefly luciferase (including hexokinase, polynucleotide kinase, T4 DNA ligase, and T4 RNA ligase) ATP could be replaced with dATP, contradicting previous data. Considering the replaceable nucleotides, not only kinases (low stringency as to ATP-requirement) but also other enzymes (moderate stringency) were typed as phosphate-directed ATP recognition. Through this study, an exact view of ATP-requiring enzymes which have a profound influence on the concentration in a cell of ATP, a metabolic and regulative key substance, was obtained, and a technically useful, fluorescent ATP-substitute (2AP-TP) was introduced.